A major sorting station in the secretory pathway is the trans Golgi network (TGN), where proteins are directed to the plasma membrane, endosomes, and lysosomes. Defects in sorting at the TGN can lead to inherited human diseases such as I-cell disease and Hermansky-Pudlak syndrome, and likely to contribute to more common, multigenic diseases such as cancer and heart disease. The long-term goal of this project is to define the molecular basis of protein sorting into vesicle-mediated pathways between the TGN and endosomes. Studies of Saccharomyces cerevisiae indicate that clathrin coated vesicles (ccv) participate in evolutionarily conserved protein transport between the TGN and endosomes. Two types of clathrin adaptors are implicated in these pathways, the AP-1 complex and monomeric Gga proteins. Analyses of the yeast adaptors has opened unique avenues to address ccv formation at the TGN/endosomes. A combination of genetic, molecular, biochemical, and cell biological strategies will be applied to achieve three specific aims. First, roles of Gga proteins and AP-1 in TGN/endosome clathrin-mediated trafficking pathways will be determined. Towards this end, roles for each adaptor in clathrin recruitment to membranes will be evaluated. Also, the relative distribution and function of each adaptor in different trafficking pathways between the TGN and endosomes will be defined. Second, the functions of two novel Gga2p-interacting proteins, Ent3p and Ent5p, will be determined. These proteins, which are required for clathrin-mediated traffic between the TGN and endosomes, are distinguished by the presence of ENTH-related domains. Function of these proteins in clathrin coat assembly and clathrin-mediated transport will be characterized in vivo using mutant cells and in vitro by development of a coat assembly assay using pure proteins and liposomes. Emphasis will be directed towards understanding the role of phosphoinositide binding by Ent3p and Ent5p ENTH-related domains. Additional TGN/endosome clathrin coat components will be identified using interaction and genetic approaches. Third, we will initiate a chemical genetic strategy to study AP-1 and Gga-mediated traffic by identifying and characterizing small molecule inhibitors. Together these studies are expected to provide significant advances in our understanding of the fundamental process of ccv formation and protein sorting at the TGN and endosomes.